The invention relates to a chromoionophore comprising an chromophore and an ionophore capable of selectively binding potassium ions for determining potassium ion in a sample. The present invention also relates to a method of determining the concentration of potassium ions in a sample wherein the chromoionophore is contacted with potassium ion in a sample, wherein the intensity of at least one absorption maximum in the visible region changes and the concentration of potassium ion is calculated based on the change in the intensity of the absorption maximum.
The accurate measurement of physiologic cations, such as sodium, potassium, lithium, calcium, and magnesium, is essential in clinical diagnosis. Traditionally, these ions were determined in plasma or serum using ion-selective electrodes (ISE), which are very cumbersome to use and costly to maintain. Serious drawbacks of electrochemical measuring arrangements are the requirement of a reference element, sensitivity towards electrical potentials and electromagnetic interference.
U.S. Pat. No. 4,367,072 describes a process for the determination of metal ions using simple crown ethers as ion-binding units. However, the binding is too weak to be useful for many practical applications, such as clinical applications, in which the indicator has to discriminate between ions with very similar properties, e.g., sodium versus potassium or magnesium versus calcium.
U.S. Pat. No. 5,011,924 and U.S. Pat. No. 4,994,395 describe cryptands (or cryptohemispherands) linked with an ionizable chromophore, which changes its color upon binding of ions based on charge interaction between the bound cation and the anion of chromophore. Although all nitrogen atoms in these cryptands are aliphatic, and not electronically conjugated with the chromophore, the results of measurement of serum samples using these chromoionophores are impressive and promising (Helgeson et. al. J. Am. Chem. Soc., vol. 111, 1989, 6339-6350). However, the syntheses of these cryptands, especially of those cryptohemispherands, are lengthy and tedious. Consequently, the manufacturing cost of these reagents remains prohibitively high even in the decades following their discovery. The cost factor could be a reason why these reagents have not replaced those ISE modules in most large clinical analyzers, in which the ISE methods are still dominating (see Burtis et. al. ed. “Tietz Textbook of Clinical chemistry and Molecular Diagnostics” Elsevier Sauders, St. Louis, Mo., USA 2006, page 986).
U.S. Pat. No. 6,211,359 (which is hereby incorporated by reference in its entirety) reports ionophores for potassium, which have π-electron conjugated nitrogen and were coupled to a fluorophore to make luminophore-ionophore sensors where the respective ions are detected by measuring luminescence emission. This ionophore has been proven to be very selective in determination of potassium in whole blood (see He et. al. Anal. Chem. Vol. 75, 2003, 449-555; and J. Am. Chem. Soc. vol. 125, 2003, 1468-1469), thus showing that the ionophores are effective at physiological pH.
By coupling to a chromophoric moiety, these ionophores can be converted into colorimetric sensors. The chromophoric moieties can be a nitro-substituted styryl or phenylazo, substituted thiazolevinyl or thiazoleazo, substituted naphthothiazolevinyl or naphthothiazoleazo, substituted naphthylvinyl or naphthylazo, substituted quinolinovinyl or quinolinoazo and their quarternized salts. To date, there has been no systematic investigation of these types of colorimetric reagents. The water solubility of the reagents can be improved dramatically if a charge is introduced into the dye molecules. The absorption wavelength can be red-shifted by replacing the nitrophenyl with a nitrothiazole or larger chromophore-generating substituent.
The present invention provides chromoionophores that are water soluble and can be reliably used for detection of ions in samples that absorb at wavelengths longer than about 400 nm. Examples of such samples are biological fluids.
For the chromoionophores of the present invention, the amount of ion present is determined by measuring changes in the intensity of at least one absorption maximum of the chromoionophore upon contacting the chromoionophore with an ion. The measurements are done by using standard centralized instruments, such as ultraviolet-visible spectrometers. A calibration curve for an ion is generated from a series of empirically determined absorption spectra. A calibration curve is useful for at-once determining the concentration of ion in a sample from the measured absorbance.
The chromoionophores of this invention absorb visible light (about 400 nm or greater) with reasonable extinction coefficient, thus avoiding those practical problems associated with variable background absorption from optical components, cuvette polymer materials, and biological samples. Further, the invention is well suited for practice in the determination of potassium ion in the presence of physiological concentrations of other alkali ions.